Over the last three decades, there has been a striking rise in obesity, together with parallel increases in insulin resistance, type-2 diabetes, cardiovascular disease and certain cancers. A critical link between obesity and its downstream comorbidities is chronic low-grade inflammation promoted by secretion of pro-inflammatory mediators by ?angry? adipocytes and macrophages in visceral adipose tissue (VAT). Anti-inflammatory cells and molecules capable of reining in these processes remain poorly understood. In 2009, we reported a population of Foxp3+CD4+ regulatory T cells (Tregs) that is highly enriched in epididymal VAT (eVAT), but not in lymphoid or other nonlymphoid organs, of lean mice as they age. These cells have a distinct gene- expression profile, T cell receptor (TCR) repertoire and profile of mediator dependencies. eVAT Tregs are strikingly and specifically reduced in several insulin-resistant mouse models of obesity, and loss- and gain-of- function experiments have confirmed that they regulate local and systemic inflammation and metabolism. An analogous Treg population is present in human omentum. eVAT Tregs serve as a paradigm for Treg populations specifically adapted to survive and function within particular tissue environments. Over the last funding cycle, we made substantial strides in illuminating the biology of VAT Tregs: dissecting transcriptome modulation with age and diet; discovering a two-step, two-site scenario of diversification from lymphoid-organ Tregs; uncovering modes of molecular diversification, including the construction of a tissue- Treg transcription-factor network; and demonstrating dependencies on TCR specificity, Foxp3 and IL-33. Building on these findings, the overall goal of this proposed project is to elucidate newly uncovered cellular and molecular elements controlling the generation, homeostasis or function of eVAT Tregs. We will undertake three Specific Aims, each designed to address a hypothesis that emerged from results obtained during the last funding cycle. 1) To examine the precursor potential of the splenic PPAR?lo Treg compartment. 2) To identify factors that elicit, promote or guide the splenic PPAR?lo precursors of eVAT PPAR?hi Tregs. 3) To determine whether and how the homeostasis and function of eVAT Tregs are influenced by a circadian clock. These studies should yield important new information on cellular and molecular pathways involved in regulating the devastating downstream consequences of obesity. Identification of novel therapeutic targets within these pathways is especially important given the disconcerting increases in these disorders and the unacceptable side-effects of certain drug options.